DESCRIPTION: (Applicant's Abstract) The goal of this program is to elucidate some critical aspects of Ca2+ signaling in astrocytes. The Na/Ca exchanger and high ouabain affinity isoforms (alpha2/alpha3) of the Na+ pump catalytic (alpha) subunit colocalize to plasma membrane (PM) microdomains that overlie "junctional" endoplasmic reticulum (jER) in astrocytes and neurons. This has fostered the hypothesis that the alpha2/alpha3 Na+ pumps help modulate Ca2+ signaling by regulating jER Ca2+ stores. To examine these ideas and test the hypothesis, the following specific aims are proposed: 1. To determine the functional organization of astrocyte ER Ca2+ stores: How are the inositol trisphosphate and ryanodine receptors (IP3R and RYR) and the ER Ca2+ pumps distributed in the ER? And, how does this distribution influence local and "global" cell Ca2+ signaling? High resolution Ca2+ imaging will be used to elucidate the sites of origin and mechanism(s) of propagation of agonist-evoked Ca2+ signals. PCR and immunoblotting will be used to identify the specific Ca2+ pumps, and IP3R and RYR isoforms expressed in primary cultured rat brain astrocytes. High spatial resolution immunocytochemistry will then be used to determine the specific locations of these pumps and receptors in the ER of cells in which Ca2+ signaling has been studied, to elucidate the relationship between transporter location and the sites of signal initiation and pathways of signal propagation. 2. To confirm that the different Na+ pump alpha subunits are differently distributed in astrocytes, and to determine how the isoforms are correctly sorted. Adenovirus transfection, a transgenic mouse model, and immunocytochemical methods, will be used to determine the specific distributions of these Na+ pump isoforms. Domain swapping with adenovirus transfection methods will be used to identify the region(s) of the Na+ pump alpha subunit isoforms that are responsible for targeting these isoforms to their specific locations on the PM. 3. To test the hypothesis that the high ouabain affinity Na+ pump isoforms and the Na/Ca exchanger, which colocalize to PM microdomains adjacent to the jER, play key roles in Ca2+ signaling. Digital imaging will be used to determine the effects of low-dose ouabain (which blocks only the alpha2/alpha3 Na+ pump isoforms) on the cytosolic Na+ concentration ([Na+]CYT; measured with SBFI) and on Ca2+ signaling (measured with Fura-2 and Furaptra). Astrocytes from normal animals, and from transgenic mice with null mutations for the Na+ pump alpha1 and alpha2 genes will be studied. The results will provide new insight into a fundamental mechanism of Ca2+ signal modulation that may also, during ischemia/anoxia, contribute to Ca2+ overload, excitotoxic injury and cell death.